Method, Kit, and Device for Preparing Glycan from Glycoprotein

ABSTRACT

Provided are a method for preparing a glycan from a glycoprotein, and a kit and a device for preparing a glycan from a glycoprotein. The method includes (I) a step of obtaining a glycan-containing sample by bringing a glycan releasing solution that contains a hydroxylamine compound and a basic reagent into contact with the glycoprotein and releasing the glycan from the glycoprotein; (II) a step of adsorbing a glycan having a length of a monosaccharide or more on a purifying agent for purifying the glycan by bringing the glycan-containing sample into contact with the purifying agent, which is composed of a compound having a betaine structure; and (III) a step of eluting the glycan from the purifying agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of InternationalApplication No. PCT/JP2019/048323 filed Dec. 10, 2019, and claimspriority to Japanese Patent Application No. 2018-233837 filed Dec. 13,2018, the disclosures of which are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to a method, a kit, and a device forpreparing a glycan from a glycoprotein.

BACKGROUND ART

Most of the proteins constituting an organism are glycosylated, and arepresent as glycoproteins to which glycans are linked. With regard toproteins, higher-order structure formation, functions such as cell-cellsignal transduction and molecular recognition, and in vivo kinetics areregulated through this glycosylation. It is known that the structure anddistribution of glycans in a glycoprotein are associated with thefunctional expression of the protein, and the glycan structure changesalong with the development and progression of many diseases, and it ispossible to use glycans of glycoproteins as biomarkers for diseases.Also, it has been suggested that glycans affect not only the activity ofbiopharmaceuticals but also the antigenicity and kinetics thereof.

Therefore, it is expected that glycoprotein structural analysis plays animportant role in various technical fields such as life science, medicalcare, and drug discovery, to elucidate mechanisms of the development ofvarious diseases associated with structural changes in glycans, todevelop disease treatment and diagnostic techniques, and the like, andthe importance of structural analysis of glycans is increasing.

Therefore, there is demand for constructing techniques for analyzing thestructures of glycans quickly, easily, and highly accurately. Glycansare usually analyzed through high-performance liquid chromatography(HPLC), nuclear magnetic resonance spectroscopy, capillaryelectrophoresis (CE method), or mass spectrometry, or a combinationthereof. However, in order to analyze glycans using the above-describedmethod, it is necessary to release the glycans from a glycoprotein, andto purify (collect) only the resulting free glycans.

Glycoproteins are classified into an N-linked glycan and an O-linkedglycan according to the binding form of glycans to proteins.Conventionally, methods that are suitable for the corresponding types ofglycans have been used to release glycans from glycoproteins

In order to release N-linked glycans, enzymes such as PNGase F andglycopeptidase A can be used, for example. However, if an enzyme is usedto release glycans, proteins are treated with a reducing reagent such asdithiothreitol in advance, are denatured through treatment using analkylating agent such as iodoacetamide, and are decomposed into peptidesusing protease such as trypsin, and then glycans are released through areaction with a glycan-releasing enzyme. Therefore, it takes a long timeto release the glycans because pretreatment needs to be performed.Furthermore, PNGase F and glycopeptidase A are very expensive and thusit is considerably costly to analyze many specimens.

Also, a method for releasing glycans through a chemical reaction such ashydrazinolysis may be used without using enzymes. Hydrazinolysis ismainly used as a method for releasing N-linked glycans, in which asufficiently dried glycoprotein is dissolved in anhydrous hydrazine, andis treated through heating at 100° C. for 10 hours or more, for example.After the reaction, anhydrous hydrazine needs to be distilled off undervacuum pressure, and acetylation needs to be performed using sodiumhydrogen carbonate and acetic anhydride. This is because hydrazine alsodecomposes and converts an N-acetyl group and an N-glycolyl group linkedto glycans to amino groups, and thus acetylation is performed to returnthem to the original groups. However, hydrazinolysis needs to beperformed under completely anhydrous conditions, and if even a smallamount of water is mixed in a reaction system, the yield willsignificantly decrease. Therefore, the reaction needs to be performedafter a sample is sufficiently dried under vacuum pressure in advance.Furthermore, the reaction time is 10 hours or longer, and, if hydrazineis distilled off after the reaction and re-acetylation is performed, ittakes at least two days or more to complete all the processes. Also,hydrazine is carcinogenic toxin and an explosive compound, and thusneeds to be handled with extreme care. Furthermore, if other acyl groupssuch as an N-glycolyl group are originally bound thereto, all of themare acetylated and also analyzed as N-acetyl form, and thus there isalso a problem in that N-glycolylneuraminic acid cannot be analyzed. Inparticular, the presence or absence of N-glycolylneuraminic acid, whichis one type of sialic acid, may be problematic in biopharmaceuticals andglycan disease markers.

Because no practical enzyme for releasing O-linked glycans has beenfound, O-linked glycans are mostly released through chemical reaction. Amethod in which glycans are subjected to β-elimination in an aqueoussolution of a strong alkali is widely used as a chemical reaction forreleasing O-linked glycans. However, the free glycans are immediatelydecomposed in the presence of alkali, and thus, a method in which thefree glycans are immediately reduced to alditol through a rection in thecoexistence of sodium borohydride is usually used.

The free alditol is an alcohol obtained by reducing an aldehyde group onthe glycan side, which serves as a functional group on which afluorescent label is to be provided, and thus no fluorescent label canbe provided. Therefore, usually, all the hydroxy groups of alditol aremethylated (complete methylation), and then mass spectrometry isperformed thereon.

The above-described hydrazinolysis is known as another method forchemically releasing O-linked glycans while preserving the aldehydegroup. After the reaction, hydrazine is distilled off, re-acetylation isperformed, a fluorescent label is provided, and then analysis isperformed through HPLC or the like.

On the other hand, in both cases where an O-linked glycan is releasedthrough hydrazinolysis and an O-linked glycan is released through alkaliβ-elimination, a side reaction (peeling reaction) always occurs in whicha sugar linked to the 3-position of N-acetylgalactosamine at thereducing end of a glycan is eliminated. There has been a problem that,even if β-elimination is performed using a weak base in order tosuppress this reaction, not only peeling is unavoidable but also releaseefficiency is significantly reduced. Furthermore, the reaction time is16 hours or longer.

Also, with a method in which sodium borohydride is used together tosuppress peeling, a labeling reagent cannot be provided to the reducingend, and thus it is not possible to perform highly sensitive analysisthrough HPLC or the like.

The inventors of the present invention have constructed a method forreleasing glycans from glycoproteins using a basic catalyst in anaqueous solution in the presence of hydroxylamine as a method forreleasing N-linked glycans and O-linked glycans from proteins using asafe and inexpensive chemicals in a short treatment time without using aspecial system (Patent Document 1).

Also, conventionally, glycans released from glycoproteins have beenpurified using a method in which hydrophilic interaction is utilized, amethod in which covalent bond formation is utilized using phenylboronicacid, a method in which interaction with lectins is utilized, a methodin which chelate interaction with titanium (e.g., titanium oxide(TiO₂)), zirconium, silver, or the like is utilized, or the like, forexample (e.g., see Non-Patent Document 1 etc.).

Here, the physicochemical properties of glycans are utilized in themethod in which hydrophilic interaction is utilized, and glycans areselectively adsorbed and collected using Sepharose beads, utilizinghydrogen bonding formed between hydroxy groups of Sepharose and glycans(e.g., see Non-Patent Documents 1 and 2 etc.). However, the interactionbetween Sepharose and glycans is weak, and thus, O-linked glycans with asmall molecular weight are influenced by impurities such as proteins andpeptide fragments contained in a sample, and as a result, the O-linkedglycans will be weakly carried by Sepharose beads. Therefore, there is aproblem that the efficiency of concentrating glycans and thereproducibility thereof are reduced.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: WO 2008/062167

Non-Patent Documents

Non-Patent Document 1: Chen-Chun Chen et al., “Interaction modes andapproaches to glycopeptide and glycoprotein enrichment”, Analyst, 2014,vol. 139, p. 688-704

Non-Patent Document 2: Michiko Tajiri et al., “Differential analysis ofsite-specific glycans on plasma and cellular fibronectins: Applicationof a hydrophilic affinity method for glycopeptide enrichment”,Glycobiology, 2005, vol. 15, no. 12, p. 1332-1340

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

In view of this, the present invention provides a glycan preparationtechnique by which a glycan can be prepared from a glycoprotein in ashort period of time through a simple operation while the decompositionof the glycan is suppressed. In particular, an object thereof is toprovide a glycan preparation technique by which glycans includingO-linked glycans with a small molecular weight can be collected in ahigh yield.

Means for Solving Problem

As a result of conducting intensive studies in order to solve theabove-described problems, the inventors of the present invention foundthat it is possible to collect glycans including O-linked glycans with asmall molecular weight in a high yield by bringing a glycan releasingsolution containing a hydroxylamine compound and a basic reagent intocontact with a glycoprotein, releasing glycans from the glycoprotein,and bringing the resulting mixture into contact with a purifying agentmade of a compound having a betaine structure. Also, the inventors foundthat this reaction can be carried out in a short period of time with asimple operation, and it is also possible to suppress decomposition(peeling) of glycans.

That is, the present invention provides a method, a kit, and a devicefor preparing a glycan from a glycoprotein, and specifically, includesthe following configurations.

(1) A method for preparing a glycan from a glycoprotein includes:

(I) a step of obtaining a glycan-containing sample that contains aglycan by bringing a glycan releasing solution that contains ahydroxylamine compound and a basic reagent into contact with theglycoprotein and releasing the glycan from the glycoprotein;

(II) a step of adsorbing a glycan having a length of a monosaccharide ormore on a purifying agent for purifying the glycan by bringing theglycan-containing sample into contact with the purifying agent, which iscomposed of a compound having a betaine structure; and

(III) a step of eluting the glycan from the purifying agent.

(2) The method according to (1) above further includes (IV) a glycanlabeling step of reacting a glycan labeling reagent in a glycan labelingsolution and the glycan with each other.

(3) The method according to (2) above further includes (V) a reductionstep of causing a reaction with a reduction solution that contains areducing reagent.

(4) In the method according to (3) above, a concentration of thereducing reagent in the reduction solution is 1.0 mmol/L or more and 250mmol/L or less.

(5) In the method according to (1) to (4) above, the glycan releasingsolution contains hydroxylamines in an amount of 2% or more and 70% orless.

(6) In the method according to (1) to (5) above, the hydroxylaminecompound is at least one selected from the group consisting ofhydroxylamine, salts of hydroxylamine, O-substituted hydroxylamine, andsalts of O-substituted hydroxylamine.

(7) In the method according to (1) to (6) above, the basic reagent is atleast one selected from the group consisting of alkali metal hydroxides,weak acid salts of alkali metals, alkaline earth metal hydroxides,alkaline earth metal salts dissolved in aqueous ammonia, and organicbases.

(8) In the method according to (7), the alkali metal hydroxide islithium hydroxide, sodium hydroxide, or potassium hydroxide, the weakacid salt of alkali metals is sodium bicarbonate or sodium carbonate,the alkaline earth metal hydroxide is calcium hydroxide, bariumhydroxide, or strontium hydroxide, the alkaline earth metal salt iscalcium acetate, calcium chloride, barium acetate, or magnesium acetate,and the organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene,1,5,7-triazabicydo[4.4.0]dec-5-ene,7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene,1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetramethylguanidine,or cetyltrimethylammonium hydroxide.

(9) In the method according to (1) to (8) above, the purifying agentcontains the compound having the betaine structure as an activeingredient.

(10) In the method according to (9) above, the purifying agent is acarrier for concentrating a glycan in which a polymer whose side chainthat has the betaine structure is bonded to a main chain thereof isimmobilized on an insoluble support.

According to the above-described configurations, glycans can be releasedfrom a glycoprotein using a safe and unexpensive chemical in a shorttreatment time while suppressing the decomposition (peeling) of theglycan. Furthermore, when a purifying agent having effectively enhancedhydrophilicity is used, the free glycans that include O-linked glycanswith a small molecular weight can be specifically adsorbed on thepurifying agent. Accordingly, it is possible to efficiently collectglycans from the glycoprotein. Therefore, according to theabove-described configurations, it is possible to prepare glycans from aglycoprotein through a simple operation in a short period of time in ahigh yield.

Also, with the configuration according to (2) above, the free glycanscan be labeled by a labeling reagent. Furthermore, with theconfiguration according to (3) above, it is possible to reduce a complexof a glycan and a labeling reagent using a reducing reagent, andaccordingly, to stabilize the complex between the glycan and thelabeling reagent and to improve the labeling efficiency. In particular,it is possible to further improve the labeling efficiency of glycans bysetting the concentration of the reducing reagent to the concentrationrange in the configuration according to (4) above.

(11) A kit for preparing a glycan from a glycoprotein includes:

(a) a hydroxylamine compound;

(b) a basic reagent; and

(c) a purifying agent for preparing a glycan having a length of amonosaccharide or more, the purifying agent being composed of a compoundhaving a betaine structure.

(12) The kit according to (11) above further includes (d) a glycanlabeling reagent.

(13) In the kit according to (11) or (12) above, the hydroxylaminecompound is at least one selected from the group consisting ofhydroxylamine, salts of hydroxylamine, O-substituted hydroxylamine, andsalts of O-substituted hydroxylamine.

(14) In the kit according to any of claims (11) to (13), the basicreagent is at least one selected from the group consisting of alkalimetal hydroxides, weak acid salts of alkali metals, alkaline earth metalhydroxides, alkaline earth metal salts dissolved in aqueous ammonia, andorganic bases.

(15) In the kit according to (14) above, the alkali metal hydroxide islithium hydroxide, sodium hydroxide, or potassium hydroxide, the weakacid salt of alkali metals is sodium bicarbonate or sodium carbonate,the alkaline earth metal hydroxide is calcium hydroxide, bariumhydroxide, or strontium hydroxide, the alkaline earth metal salt iscalcium acetate, calcium chloride, barium acetate, or magnesium acetate,and the organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene,1,5,7-Triazabicydo[4.4.0]dec-5-ene,7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene,1,1,3,3-Tetramethylguanidine, 2-tert-Butyl-1,1,3,3-tetramethylguanidine,or cetyltrimethylammonium hydroxide.

According to the above-described configurations, a glycan can beprepared more easily and in a short period of time by assemblingreagents required for preparing the glycan from a glycoprotein into akit.

(16) A device for preparing a glycan from a glycoprotein includes:

a first container holding portion configured to hold a first containerin which a glycoprotein-containing sample that contains the glycoproteinis accommodated; a reagent introducing portion configured to introduce areagent into the first container; and a second container holding portionconfigured to hold a second container that includes a purifying agentfor purifying a glycan having a length of a monosaccharide or more, thepurifying agent being composed of a compound having a betaine structure;

in which the reagent introducing portion includes a glycan releasingreagent introducing portion configured to introduce a glycan releasingreagent that contains a hydroxylamine compound and a basic reagent intothe first container.

(17) In the device according to (16) above, the reagent introducingportion further includes a glycan labeling reagent introducing portionconfigured to introduce a glycan labeling reagent.

(18) The device according to (16) or (17) above further includes asolid-liquid separation portion configured to subject a content of thesecond container to solid- liquid separation.

According to the above-described configurations, a glycan can beprepared more easily and in a short period of time by assemblingreagents and members required for preparing the glycan from aglycoprotein into a device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram schematically showing an example of a glycanpreparing and purifying agent according to an embodiment.

FIG. 2 is a graph showing the results of Example 2 obtained whenexamining the relationship between the concentration of a reducingreagent at the time of glycan labeling and the yield (on the lowconcentration side).

FIG. 3 is a graph showing the results of Example 2 obtained whenexamining the relationship between the concentration of a reducingreagent at the time of glycan labeling and the yield (on the highconcentration side).

FIG. 4 is a chart of HPLC analysis showing the results of Example 3obtained when examining the preparation of glycans, through a comparisonbetween Comparative Example 4 and Comparative Example 5.

FIG. 5 is a graph showing the results of Example 3 obtained whenexamining the preparation of glycans, through a comparison betweenComparative Example 4 and Comparative Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes embodiments of the present invention in detail.However, the present invention is not limited to the embodiments, whichwill be described later.

Method for Preparing Glycan from Glycoprotein

A method for preparing a glycan from a glycoprotein according to thisembodiment includes:

(I) a step of obtaining a glycan-containing sample that contains aglycan by bringing a glycan releasing solution that contains ahydroxylamine compound and a basic reagent into contact with theglycoprotein and releasing the glycan from the glycoprotein;

(II) a step of adsorbing a glycan having a length of a monosaccharide ormore on a purifying agent for purifying the glycan by bringing theglycan-containing sample into contact with the purifying agent, which iscomposed of a compound having a betaine structure; and

(III) a step of eluting the glycan from the purifying agent, and thusthe glycan can be prepared from the glycoprotein.

In this specification, a “glycoprotein” refers to a protein in which atleast one or more O-linked glycans or N-linked glycans are linked to anamino acid sequence of the protein. There is no particular limitation onthe glycoprotein targeted for the method for preparing a glycan from aglycoprotein, and the glycoprotein may be naturally derived orsynthesized.

Also, a “glycan” may include an O-linked glycan and an N-linked glycan,and both the O-linked glycan and the N-linked glycan can be preparedfrom a glycoprotein. The O-linked glycan has a structure in which aglycan is linked to a side chain of an amino acid residue serine (Ser)or threonine (Thr) in a protein via an —OH group included in the aminoacid side chain. Also, the O-linked glycans are classified into one toeight types according to the core structure. Also, the N-linked glycanrefers to a glycan that is linked to a nitrogen atom of an amide groupin a side chain of an asparagine residue of a protein. The N-linkedglycans include glycans that form branches with mannose used as a basepoint, and examples thereof include two branched, three branched, andfour branched glycans, and the like. Also, the N-linked glycans can beclassified into basic, high mannose, hybrid, and complex types, and thelike according to the structures thereof.

Step (I)/Glycan-Containing Sample Acquisition Step

Step (I) is a step of obtaining a glycan-containing sample that containsa glycan by bringing a glycan releasing solution containing ahydroxylamine compound and a basic reagent into contact with theglycoprotein and releasing the glycan from the glycoprotein.

There is no limitation on the order of the contact with a glycoproteinand mixing operations as long as a “glycan releasing solution” finallyreaches a state in which the glycoprotein, hydroxylamines, and a basicreagent are in contact with each other. Hydroxylamines may first beadded to a glycoprotein, and a basic reagent may be added thereto, forexample. Alternatively, a basic reagent may first be added to aglycoprotein, and hydroxylamines may be added thereto. Alternatively,hydroxylamines and a basic reagent may first be mixed together, and aglycoprotein may be added thereto. Note that, in the case of O-linkedglycans, it is preferable to first add hydroxylamines in order tosuppress decomposition (peeling) of a glycan.

Here, examples of “hydroxylamines” that can be used for this embodimentinclude hydroxylamine, salts of hydroxylamine, O-substitutedhydroxylamine, and salts of 0-substituted hydroxylamine. Specifically,examples thereof include, but are not limited to, at least one compoundselected from the group consisting of hydroxylamine hydrochloride, anaqueous solution of hydroxylamine, hydroxylamine sulfate, hydroxylaminephosphate, O-methylhydroxylamine hydrochloride, O-ethylhydroxylaminehydrochloride, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, nitrobenzylhydroxylamine hydrochloride, O-(tert-butyldimethylsilyl)hydroxylamine,and O-(trimethylsilyl)hydroxylamine. Note that the above-describedcompounds may be used in combination of two or more. In a preferredembodiment, “hydroxylamines” are aqueous solutions of hydroxylamine.

The final concentration of “hydroxylamines” may be set to aconcentration range of 2% (w/w) or more and 70% (w/w) or less, forexample, and to a concentration range of 2% (w/w) or more and 50% (w/w)or less, for example. However, the final concentration thereof is notlimited to the above-described concentration range, and can be adjustedby those skilled in the art as appropriate depending on the type oftarget glycoprotein, other components (amines, a basic reagent, andother additive agents), contact conditions (time, temperature, etc.),and the like.

In particular, the final concentration of “hydroxylamines” is preferablyas high as possible in order to release O-linked glycans. Therefore, inorder to release O-linked glycans, the final concentration of“hydroxylamines” may be set to a concentration range of 5% (w/w) or moreand 70% (w/w) or less, for example, and to a concentration range of 10%(w/w) or more and 60% (w/w) or less, for example.

Note that, in order to release N-linked glycans, the final concentrationof “hydroxylamines” may be set to a concentration range of 2% (w/w) ormore and 50% (w/w) or less, and may be preferably set to a concentrationrange of 2% (w/w) or more and 20% (w/w) or less.

When hydroxylamine is used as “hydroxylamines”, if the concentration ofhydroxylamine is set to more than 2% (w/w) and 50% (w/w) or less, asufficient amount of glycan can be collected, and hydroxylamine islikely to be stable.

Also, examples of the “basic reagent” that can be used in thisembodiment include at least one compound selected from the groupconsisting of alkali metal hydroxides, weak acid salts of alkali metals,alkaline earth metal hydroxides, alkaline earth metal salts dissolved inaqueous ammonia, and organic bases.

Examples of alkali metal hydroxides are not limited to the following,and include lithium hydroxide, sodium hydroxide, and potassiumhydroxide.

Also, examples of weak acid salts of alkali metals are not limited tothe following, and include sodium bicarbonate and sodium carbonate.

Also, examples of alkaline earth metal hydroxides are not limited to thefollowing, and include calcium hydroxide, barium hydroxide, andstrontium hydroxide.

Also, examples of alkaline earth metal salts dissolved in aqueousammonia are not limited to the following, and include calcium acetate,calcium chloride, barium acetate, and magnesium acetate.

In particular, lithium hydroxide is preferable.

Also, examples of organic bases are not limited to the following, andinclude DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene, TBD:1,5,7-Triazabicyclo[4.4.0]dec-5-ene, MTBD:7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, TMG:1,1,3,3-Tetramethylguanidine, t-BuTMG: 2-tert-Butyl-1,1,3,3-tetramethylguanidine), DBN:1,5-diazabicydo[4.3.0]non-5-ene, and CTAH: cetyltrimethylammoniumhydroxide. Note that the above-described compounds may be used incombination of two or more. In a preferred embodiment, an “organic base”is an organic strong base (with a pK_(a) value of 12 or more), andspecific examples thereof include DBU, TMG, TBD, MTBD, and CTAH. TheseDBU, TMG, TBD, MTBD, and CTAH are preferably used as organic basesbecause the bases can be removed after reaction through washing with anorganic solvent

The final concentration of “basic reagent” may be set to a concentrationrange of 2 mM or more and 10 M or less, for example. However, the finalconcentration thereof is not limited to the above-describedconcentration range, and can be adjusted by those skilled in the art asappropriate depending on the type of target glycoprotein, othercomponents (hydroxylamines and other additive agents) in a reactionsolution, reaction conditions (time, temperature, etc.), and the like.Note that, when lithium hydroxide is used as a “basic reagent”, thefinal concentration thereof may be set to 6 mM or more and 8 M or less,for example. The reaction time can be shortened by setting theconcentration of basic reagent to the above-described lower limit ormore, and the influence of the remaining basic reagent on the subsequentprocess can be suppressed by setting the concentration of basic reagentto the above-described upper limit or less.

The mole ratio between hydroxylamines and a basic reagent is preferablyset to 1:2 or more and 300:1 or less, and more preferably set to 1:1 ormore and 100:1 or less. By setting the mole ratio between hydroxylaminesand the basic reagent to the above-described range, it is possible tosuppress decomposition (peeling) reaction of free glycan and to improvethe yield of glycans.

There is no particular limitation on conditions (such as the temperatureand the time) under which glycoproteins and a glycan releasing solutionare contact with each other as long as glycans can be released from atarget protein, and the conditions can be set by those skilled in theart as appropriate depending on the conditions such as the types andconcentrations of the target glycoprotein, hydroxylamines, and basicreagent and the like. Note that the temperature may be set to a range ofroom temperature or higher and 80° C. or lower, for example. Note thatglycan decomposition (peeling) rate can be reduced by lowering thereaction temperature. In particular, an N-glycolyl group and the likeare likely to be decomposed in reaction at a high temperature.Therefore, if a target protein is a glycoprotein having an unknownglycan, the reaction temperature is preferably set to 50° C. or less,and may be set to about 37° C., for example. Also, the time may be setto about 5 minutes to 16 hours depending on conditions.

Amines may be further added to the glycan releasing solution. Examplesof amines are not limited to the following, and may include at least onecompound selected from the group consisting of aqueous ammonia, anaqueous solution of methylamine, an aqueous solution of dimethylamine,ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine,morpholine, DABCO, and anthranilic acid. Furthermore, note that theabove-described compounds may be used in combination of two or more.Preferably, amines are aqueous ammonia, morpholine, DABCO, andanthranilic acid. Aqueous ammonia, morpholine, DABCO, and anthranilicacid are preferably used as amines because peeling, isomerization, anddecomposition of amides are suppressed. In particular, morpholine,DABCO, anthranilic acid, and the like, which have a lower pK_(a) valuethan that of ammonia, are preferably used because it is possible tosuppress isomerization, which is a side reaction occurring when N-linkedglycans are released.

The final concentration of “amines” may be set to a concentration rangeof 40 mM or more and 15 M or less, for example. However, the finalconcentration thereof is not limited to the above-describedconcentration range, and can be adjusted by those skilled in the art asappropriate depending on the type of target glycoprotein, othercomponents (hydroxylamines, a basic reagent, and other additive agents)in a reaction solution, reaction conditions (time, temperature, etc.),and the like. Note that, when aqueous ammonia is used as amines, thefinal concentration of ammonia in the reaction solution may be set to 2%(w/w) or more and 25% (w/w) or less, and may be preferably set to 10%(w/w) or more and 20% (w/w) or less. More preferably, the finalconcentration thereof may be 20% (w/w).

Here, as described above, in particular, the final concentration of“hydroxylamines” is preferably as high as possible when releasingO-linked glycans. However, if a liquid mixture of glycoproteins and thereaction solution contains hydroxylamines in a high concentration,unreacted hydroxylamines may remain in the liquid mixture obtained afterglycans are released. When glycans are labeled and analyzed, theunreacted hydroxylamines inhibit a labeling reaction, and thus it ispreferable to remove the unreacted hydroxylamines.

In view of this, the preparation method may further include anadditional step of removing unreacted hydroxylamines. Therefore, thepreparation method may further include a step of adding a ketone, analdehyde, or an acid anhydride to the glycan-containing sample toconvert hydroxylamines remaining in the glycan-containing sample to aketoxime, an aldoxime, or an amide.

Hydroxylamines can be converted to ketoximes through reaction withketones. Hydroxylamines can be converted to aldoximes through reactionwith aldehydes. Also, hydroxylamines can be converted to amides throughreaction with acid anhydrides.

Acetone, methyl ethyl ketone, methyl isobutyl ketone, 4-hydroxybutanone,and the like may be used as ketones. Also, salicylaldehyde,benzaldehyde, 4-hydroxybenzaldehyde, and the like may be used asaldehydes. Also, acetic anhydride, succinic anhydride, and the like maybe used as acid anhydrides.

Step (II)/Glycan Adsorption Step

Step (II) is a step of adsorbing a glycan having a length of amonosaccharide or more on a purifying agent for purifying the glycan bybringing the glycan-containing sample obtained in Step (I): theglycan-containing sample acquisition step into contact with thepurifying agent, which is composed of a compound having a betainestructure. Glycans having a length of a monosaccharide or more can beefficiently adsorbed on the purifying agent.

The “purifying agent” used in this embodiment may be referred to as apurifying agent for purifying glycans with a length of monosaccharide ormore, the purifying agent containing a compound having a betainestructure as an active ingredient. That is, the “purifying agent” may bein the form of mixture with a compound that does not have a betainestructure as long as the purifying agent contains a compound having abetaine structure.

The above-described “betaine structure” may be a structure representedby Formula (1) below.

-A-L-   (1)

[where in Formula (1), Z represents a cationic group selected from thegroup consisting of a secondary amino group, a tertiary amino group, aquaternary ammonium group, and an imino group, L represents an alkylenegroup with 1 to 10 carbon atoms, and A represents an anionic groupselected from the group consisting of a phosphate group, a carboxylgroup, a phosphonate group, a phosphinate group, a sulfonate group, asulfine group, a sulfene group, a hydroxy group, a thiol group, and aboronic acid group].

The above-described compound having the betaine structure may bebetaine, or a polymer in which a side chain having a betaine structureis bonded to a main chain thereof.

The “main chain” refers to the longest carbon chain in the polymerstructure, and the structure branched from the main chain is referred toas a “side chain”.

Also, glycans include monosaccharides. Thus, a glycan having a length ofmonosaccharide or more include a glycan having a length ofmonosaccharide, disaccharide, trisaccharide, or tetrasaccharide or more.The molecular weight of a glycan having a length of monosaccharide ormore may be about 150 or more and 3000 or less, for example.

The “purifying agent” may be immobilized on an insoluble support to forma carrier for concentrating glycans. “Purification” may also be referredto as “concentration”, and a “carrier for concentrating a glycan” may besimply referred to as a “carrier”. In a “purifying agent”, a polymer maybe immobilized on an insoluble support, and a side chain having abetaine structure may be bonded to a main chain in this polymer, forexample. The hydrophilicity is extremely increased due to the side chainhaving a betaine structure, and hydrophilic interaction makes itpossible to strongly retain high hydrophilic glycans.

In the “carrier for concentrating a glycan”, a polymer is immobilized onan insoluble support, and preferably, the polymer covers the entire or aportion of the surface of the insoluble support, forming a polymerlayer. Here, “covering” refers to a polymer being attached to thesurface of the insoluble support. FIG. 1 schematically shows an exampleof the “carrier for concentrating a glycan”. The “carrier forconcentrating a glycan” shown in FIG. 1 is provided with a polymer layerhaving a betaine structure on the surface of an insoluble support.

The “polymer layer” may contain a polymer that does not have a betainestructure, in addition to the polymer whose side chain having a betainestructure is bonded to the main chain thereof. Therefore, the “carrierfor concentrating a glycan” enables the purification of a glycan havinga length of monosaccharide or more as long as the surface of thiscarrier has a betaine structure.

The “insoluble support” is a base member that is insoluble in water andan organic solvent that is to be used in a process for preparing theglycan, and is not particularly limited as long as it can immobilize apolymer whose side chain having a betaine structure is bonded to themain chain thereof, and a known base member can be used. The material ofthe insoluble support may be either an inorganic substance or an organicsubstance, or may be a composite substance in which an inorganicsubstance and an organic substance are used in combination. Examples ofthe inorganic substance include silicon compounds such as silica, glasssuch as silicate glass, oxides such as iron oxide (ferrite, magnetite,etc.), alumina, titania, and zirconia, metals such as iron, copper,gold, silver, platinum, cobalt, aluminum, palladium, iridium, andrhodium and alloys thereof, and carbon materials such as graphite. Thesemay be used alone or in combination of two or more. Examples of theorganic substance include synthetic polymers such as crosslinkedpolyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide,and crosslinked polystyrene, and polysaccharides such as crosslinkedSepharose, crystalline cellulose, crosslinked cellulose, crosslinkedamylose, crosslinked agarose, and crosslinked dextran. These may be usedalone or in combination of two or more. Also, a polymer whose side chainhaving a betaine structure is bonded to the main chain thereof may forman insoluble support.

It is preferable to use an inorganic substance as an insoluble support,and a silicon compound is more preferable, and silica is particularlypreferable. An organic substance, which may serve as an insolublesupport, usually has a specific gravity of about 1, and solid-liquidseparation is likely to be complicated due to the difference in specificgravity between the organic substance and the glycan-containing samplebeing small. When a carrier for concentrating a glycan in which aninorganic substance is employed is used to concentrate the glycan, forexample, it is possible to perform solid-liquid separation easily andsimply, and to effectively separate the glycan adsorbed on the carrierfrom impurities such as free proteins, peptide fragments, lipids, andsalts. This can contribute to improvement of the efficiency ofconcentrating glycans. Also, an inorganic substance can impartappropriate strength to a carrier.

The insoluble support may be a support having voids, such as a porousbody or a hollow body. Examples of the porous body include monolithicsilica. Monolithic silica is a silica porous structure that hasmicrometer-sized three-dimensional mesh-like pores (micropores), andnanometer-sized pores (mesopores) in a silica skeleton that forms athree-dimensional mesh-like structure. It is possible to independentlycontrol the pore size of micropores to be in a range of 1 μm or more and100 μm or less, for example, and preferably 1 μm or more and 50 μm orless, and the pore size of mesopores to be in a range of 1 nm or moreand 100 nm or less, for example, and preferably 1 nm or more and 70 nmor less. The specific surface area of the carrier for concentrating aglycan is increased by using an insoluble support having such voids, andit is possible to increase the amount of purifying agent to beimmobilized on the surface of the insoluble support. Accordingly, whenthe carrier of this embodiment is used to concentrate glycans, theefficiency of contact with the glycans can be improved, the glycans canbe efficiently adsorbed, which contributing to the improvement of theglycan concentration efficiency. Furthermore, it is also possible to usethe carrier in order to adjust the specific gravity of an insolublesupport, which will be described later.

If the purifying agent is a polymer, this polymer may be a polymer ofpolymerizable monomers. There is no particular limitation on thepolymerizable monomer as long as it can form a polymer through apolymerization reaction. The polymerizable monomer is preferably a(meth)acrylic compound having a (meth)acryloyl group, including(meth)acrylic acid ester and derivatives thereof, for example.Furthermore, examples thereof include, but are not limited to, compoundsthat have a vinyl group, an allyl group, an α-alkoxymethylacryloylgroup, a maleic acid residue, a fumaric acid residue, an itaconic acidresidue, a crotonic acid residue, an isocrotonic acid residue, acitraconic anhydride residue, and the like, and derivatives thereof. Thepolymerizable monomers may be used alone or in combination of two ormore. Note that the “(meth)acryloyl group” represents “acryloyl group”or “methacryloyl group”, and the “(meth)acrylic” represents “acrylic” or“methacrylic”.

A side chain of the polymer to be immobilized on the insoluble supportis a molecular chain branched from the main chain thereof that iscomposed of the polymer of the above-described polymerizable monomer,and some or all of them have a betaine structure. The “betainestructure” refers to a structure that has a cationic site and an anionicsite at positions where they are separated from each other and are notadjacent to each other in the same molecule.

The “cationic site” is a positively charged atomic group, and refers toa so-called cationic group. Examples of the cationic group include, butare not limited to, a primary amino group, a secondary amino group(—NHR), a tertiary amino group (—NR₂), a quaternary ammonium group (—NR₃⁺), and an imino group. “R” in the secondary amino group, the tertiaryamino group, and the quaternary ammonium group represents an alkyl groupor an aryl group. If one group has multiple numbers of R, they may bedifferent from each other or may be the same. Examples thereof include,but are not limited to, a methyl group, an ethyl group, and a propylgroup. R preferably represents a quaternary ammonium group, andparticularly preferably a trimethyl ammonium group. Also, examples ofthe cationic group include salts formed with fluoride ions, chlorideions, bromide ions, iodide ions, hydrochloric acid ions, acetate ions,sulfate ions, hydrofluoride ions, and carbonate ions.

The “anionic site” is a negatively charged atomic group, and refers to aso-called anionic group. Examples of the anionic group include, but arenot limited to, a phosphate group, a phosphonate group, a phosphinategroup, a sulfonate group, a sulfine group, a sulfene group, a carboxylgroup, a hydroxy group, a thiol group, and a boronic acid group. Thephosphate group is preferable. Furthermore, examples of the anionicgroup also include salts formed with alkali metal ions such as sodiumions and potassium ions and alkaline earth metal ions such as calciumions.

There is no particular limitation on the “betaine structure” as long asit has a structure having the above-described cationic and anionicsites, and there is no particular limitation on a combination of thecationic site and the anionic site. Preferably, the cationic site is aquaternary ammonium group, and the anionic site is a phosphate group.

There is no particular limitation on a polymer to be immobilized on aninsoluble support as long as its side chain having a betaine structureis bonded to its main chain that is composed of a polymer ofpolymerizable monomer. Therefore, the polymer may be a copolymer of apolymerizable monomer having a cationic site and a polymerizable monomerhaving an anionic site, in addition to a homopolymer of a polymerizablemonomer having a betaine structure. Also, the polymer may be a copolymercontaining a polymerizable monomer having no charge, and the solubilityof the polymer in water and the like can be controlled due to thepolymer containing such a polymerizable monomer. A copolymer refers to apolymer obtained from two or more types of monomers, and may be any oneof an alternating copolymer, a block copolymer, a random copolymer, agraft copolymer, and the like. Therefore, the betaine structure may beintroduced for each monomer unit of the polymer, may be introduced forevery certain monomer units, or may be introduced at random.

Preferably, the polymer side chain is a homopolymer of a polymerizablemonomer having a betaine structure. In this case, the anionic site andthe cationic site are present in the same molecular chain in thepolymerizable monomer having the betaine structure. There is noparticular limitation on a linker for linking them together as long asit has a divalent or higher valent group, and a known linker can beused. An alkylene linker is preferable, and examples of the alkylenelinker include alkylene linkers having 1 or more and 10 or less carbonatoms, preferably 2 or more and 5 or less carbon atoms.

Examples of such a polymerizable monomer having a betaine structureinclude, but are not limited to, a phosphobetaine-based monomer having aphosphobetaine group such as a phosphorylcholine group, acarboxybetaine-based monomer having a carboxybetaine group, and asulfobetaine-based monomer having a sulfobetaine group. Aphosphobetaine-based monomer is preferable, and in particular, aphosphobetaine-based monomer having a phosphorylcholine group ispreferable.

Examples of the polymerizable monomer having a phosphorylcholine groupas the phosphobetaine-based monomer preferably include2-(meth)acryloyloxyethyl phosphorylcholine,2-(meth)acryloyloxyethoxyethyl phosphorylcholine,6-(meth)acryloyloxyhexyl phosphorylcholine,10-(meth)acryloyloxyethoxynonyl phosphorylcholine,2-(meth)acryloyloxypropyl phosphorylcholine, and2-(meth)acryloyloxybutyl phosphorylcholine. In particular,2-(meth)acryloyloxyethyl phosphorylcholine is particularly preferabledue to its availability. Furthermore, examples of thephosphobetaine-based monomer includedimethyl(2-methacryloyloxyethyl)(2-phosphonatoethyl)aminium,dimethyl(2-acryloyloxyethyl)(2-phosphonatoethyl)aminium,dimethyl(2-methacryloyloxyethyl)(3-phosphonatopropyl)aminium,dimethyl(2-acryloyloxyethyl)(3-phosphonatopropyl)aminium,dimethyl(2-methacryloyloxyethyl)(4-phosphonatobutyl)aminium,dimethyl(2-acryloyloxyethyl)(4-phosphonatobutyl)aminium,dimethyl(2-methacryloyloxyethyl)(phosphonatomethyl)aminium, anddimethyl(2-acryloyloxyethyl)(phosphonatomethyl)aminium.

Examples of the carboxybetaine-based monomer includedimethyl(2-methacryloyloxyethyl)(2-carboxylatoethyl)aminium,dimethyl(2-acryloyloxyethyl)(2-carboxylatoethyl)aminium,dimethyl(2-methacryloyloxyethyl)(3-carboxylatopropyl)aminium,dimethyl(2-acryloyloxyethyl)(3-carboxylatopropyl)aminium,dimethyl(2-methacryloyloxyethyl)(4-carboxylatobutyl)aminium,dimethyl(2-acryloyloxyethyl)(4-carboxylatobutyl)aminium,dimethyl(2-methacryloyloxyethyl)(carboxylatomethyl)aminium, anddimethyl(2-acryloyloxyethyl)(carboxylatomethyl)aminium.

Examples of the sulfobetaine-based monomer includedimethyl(2-methacryloyloxyethyl)(2-sulfonatoethyl)aminium,dimethyl(2-acryloyloxyethyl)(2-sulfonatoethyl)aminium,dimethyl(2-methacryloyloxyethyl)(3-sulfonatopropyl)aminium,dimethyl(2-acryloyloxyethyl)(3-sulfonatopropyl)aminium,dimethyl(2-methacryloyloxyethyl)(4-sulfonatobutyl)aminium,dimethyl(2-acryloyloxyethyl)(4-sulfonatobutyl)aminium,dimethyl(2-methacryloyloxyethyl)(sulfonatomethyl)aminium, anddimethyl(2-acryloyloxyethyl)(sulfonatomethyl)aminium.

The weight of the polymer bonded to the insoluble support is preferablyabout 0.5 mg or more and 1.5 mg or less per unit surface area (m²) ofthe insoluble support, particularly preferably 0.6 mg or more and 1.3 mgor less, and even more preferably 0.7 mg or more and 1.2 mg or less.When the polymer weight per unit surface area is in the above-describedrange, the polymer can be easily handled during polymer synthesis, andit is possible to ensure good efficiency of contact with glycans andefficiently adsorb glycans.

The “carrier for concentrating a glycan” preferably has a specificgravity of about 1.05 or more and 3.00 or less, particularly preferablyhas a specific gravity of 1.1 or more and 2.7 or less, and even morepreferably has a specific gravity of 1.5 or more and 2.5 or less. Whenthe specific gravity thereof is less than the lower limit, thesedimentation properties deteriorate, and when the specific gravityexceeds the upper limit, the dispersibility deteriorate. Therefore,operability deteriorates in any cases. Therefore, if the specificgravity of the carrier according to this embodiment is in theabove-described range and this carrier is used to concentrate glycans,for example, solid-liquid separation can be performed easily and simplythrough spontaneous sedimentation by gravity, centrifugation, or thelike due to the carrier having good sedimentation properties, and it ispossible to effectively separate glycans adsorbed on the carrier andimpurities such as free proteins and peptide fragments. Also, thecontact efficiency with glycans is improved due to good dispersibility,and the glycans can be efficiently adsorbed. Therefore, it is possibleto provide a carrier having good operability, and if the carrier is usedto concentrate glycans, for example, it is possible to provide a carrierthat has good efficiency in separation from impurities such as freeproteins and peptide fragments, and has good glycan adsorptionefficiency.

The shape of “carrier for concentrating a glycan” is not particularlylimited, and the “carrier for concentrating a glycan” may have any knownshape. Examples thereof include a spherical shape such as beads, a plateshape such as a substrate or a multi-well plate, a film shape such as asheet, a film, or a membrane, and a fibrous shape. The carrier can berephrased as a solid phase. The carrier preferably has a shape thatfacilitates handling, such as a spherical shape or a shape similarthereto. If the carrier is spherical, the average particle size ispreferably about 0.5 μm or more and 100 μm or less, and particularlypreferably 1 μm or more and 50 μm or less, or preferably 1 μm or moreand 10 μm or less. Particularly preferably, the average particle sizethereof is 3 μm or more and 10 μm or less. If the average particle sizeis less than the lower limit, it is difficult to collect the carrierthrough centrifugation or filtration, and, when a column or the like isfilled with the carrier, liquid permeability will deteriorate and alarge pressure need be applied when liquid flows therethrough. On theother hand, if the average particle size exceeds the upper limit, thecontact area between the carrier and a sample solution decreases, glycanadsorption efficiency decreases, and the glycan concentration efficiencydecreases. Therefore, when the average particle size of the “carrier forconcentrating a glycan” is in the above-described range, it is possibleto provide a carrier with high operability and provide a carrier withgood operability, and, it is possible to provide a carrier that has goodefficiency in separation from free peptide fragments and the like andhas good glycan adsorption efficiency if the carrier is used toconcentrate glycans, for example. The average particle size can bemeasured using a particle size distribution meter or the like, forexample.

The “carrier for concentrating a glycan” may be used in a state in whicha filter cup of a spin column etc., wells of a multi-well plate, wellsof a filter plate, or a container such as a microtube is filled with thecarrier.

Although the polymer can be obtained by polymerizing the above-describedpolymerizable monomer, a polymer polymerization method is notparticularly limited and can be selected as appropriate according to thetype of polymerizable monomer and the like. Radical polymerization ispreferable.

The polymer may be immobilized on an insoluble support through eitherphysical adsorption or chemical bonding. From the viewpoint of safety,chemical bonding is preferable, and it is possible to suppress elutionof polymer from the insoluble support. Also, the polymer may beimmobilized on the surface of the insoluble support by polymerizing apolymerizable monomer on the surface of the insoluble support, or aprepolymerized polymer may be immobilized on the surface of theinsoluble support.

If the polymer is immobilized on the surface of the insoluble support bypolymerizing a polymerizable monomer on the surface of the insolublesupport, a polymerization initiation point may be introduced on thesurface of the insoluble support, the insoluble support provided withthe polymerization initiation point may be immersed in a polymerizablemonomer solution, and a polymerization initiator may be added to growthe polymer from the polymerization initial point, for example.Accordingly, the polymer can be immobilized on the surface of theinsoluble support through chemical bonding. A polymerizable functionalgroup, a chain transfer group, a dormant species in living radicalpolymerization, or the like can be used as the polymerization initiationpoint.

Examples of the polymerizable functional group include a vinyl group, anallyl group (2-propenyl group), a (meth)acryloyl group, an epoxy group,and a styrene group. Examples of the chain transfer group include amercapto group and an amino group, and the mercapto group is preferablebecause it has higher reactivity.

Although not particularly limited, it is preferable to use a silanecoupling agent having a polymerizable functional group or a chaintransfer group as a method for introducing a polymerizable functionalgroup or a chain transfer group on the surface of the insoluble support.

Examples of the silane coupling agent having a polymerizable functionalgroup include (3-methacryloxypropyl)dimethylmethoxysilane,(3-methacryloxypropyl)diethylmethoxysilane,(3-methacryloxypropyl)dimethylethoxysilane,(3-methacryloxypropyl)diethylethoxysilane,(3-methacryloxypropyl)methyldimethoxysilane,(3-methacryloxypropyl)ethyldimethoxysilane,(3-methacryloxypropyl)methyldiethoxysilane,(3-methacryloxypropyl)ethyldiethoxysilane,(3-methacryloxypropyl)trimethoxysilane, and(3-methacryloxypropyl)triethoxysilane. From the viewpoint of reactivityand availability, (3-methacryloxypropyl)trimethoxysilane and(3-methacryloxypropyl)triethoxysilane are preferable. These silanecoupling agents may be used alone or in combination of two or more.

Examples of the silane coupling agent having a chain transfer groupinclude (3-mercaptopropyl)trimethoxysilane,(3-mercaptopropyl)methyldimethoxysilane,(3-mercaptopropyl)dimethylmethoxysilane,(3-mercaptopropyl)triethoxysilane,(3-mercaptopropyl)methyldiethoxysilane,(3-mercaptopropyl)dimethylethoxysilane,(mercaptomethyl)trimethoxysilane, (mercaptomethyl)methyldimethoxysilane,(mercaptomethyl)dimethylmethoxysilane, (mercaptomethyl)triethoxysilane,(mercaptomethyl)methyldiethoxysilane, and(mercaptomethyl)dimethylethoxysilane, and(3-mercaptopropyl)trimethoxysilane and (3-mercaptopropyl)triethoxysilaneare preferable from the viewpoint of availability. These silane couplingagents may be used alone or in combination of two or more.

As a result of forming a covalent bond between a silane coupling agenthaving a polymerizable functional group or a chain transfer group and afunctional group on the surface of the insoluble support, thepolymerizable functional group or the chain transfer group can beintroduced onto the insoluble support using the silane coupling agent,for example. If alkoxysilanes such as trimethoxysilanes andtriethoxysilanes are used as a silane coupling agent, for example, thepolymerizable functional group or the chain transfer group can beintroduced as a result of a silanol group generated through hydrolysisand a hydroxy group, an amino group, a carbonyl group, or a silanolgroup present on the surface of the insoluble support undergoingdehydration and condensation to form a covalent bond.

A polymer layer is formed on the surface of the insoluble support byintroducing the polymerizable functional group or the chain transfergroup on the surface of the insoluble support, and then mixing theinsoluble support and the polymerizable monomer together to proceed thepolymerization reaction. Although there is no limitation on thepolymerization reaction, the polymerization reaction is performed byintroducing the insoluble support into a solvent in which apolymerizable monomer and a polymerization initiator are dissolved, andheating the mixture at a temperature of 0° C. or more and 80° C. or lessfor 1 hour or more and 30 hours or less under stirring, for example.Thereafter, the insoluble support is filtered under vacuum pressure, andis cleaned and dried.

The ratio of the used insoluble support, polymerizable monomer, andpolymerization initiator is not particularly limited, and they areusually used in a ratio of 0.1 mmol or more and 10 mmol or less of thepolymerizable monomer and 0.01 mmol or more and 10 mmol or less of thepolymerization initiator relative to 1 g of the insoluble support.

Any solvent can be used as long as the corresponding polymerizablemonomer is soluble, and examples thereof include alcohols such asmethanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, andn-pentanol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane,chloroform, cyclohexanone, N,N-dimethylformamide, dimethyl sulfoxide,methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone,methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycolmonomethyl ether, and ethylene glycol monobutyl ether. These solventsare used alone or in combination of two or more.

Examples of the polymerization initiator include, but are notparticularly limited to, azo compounds such as2,2′-azobisisobutylnitrile (may be abbreviated as “AIBN”), and1,1′-azobis(cydohexane-1-carbonitrile), organic peroxides such asbenzoyl peroxide, lauryl peroxide, and tert-butyl peroxide, and redoxinitiators such as hydrogen peroxide-ferrous iron.

On the other hand, if a prepolymerized polymer is immobilized on thesurface of the insoluble support, a method for physically adsorbing theprepolymerized polymer on the insoluble support, or a method forchemically bonding the prepolymerized polymer thereto may be used, forexample. Preferably, a component that is likely to be adsorbed on theinsoluble support, or a component that has a functional group that canreact with a reactive functional group that is present on the surface ofthe insoluble support is incorporated into a polymer as a copolymerduring the polymerization of the polymer. As the functional group thatcan react with the reactive functional group that is present on thesurface of the insoluble support, for example, a silanol group and thelike are preferable due to having high reactivity, the silanol groupbeing obtained through hydrolysis of a silane coupling agent and thelike, and the silanol group can form a covalent bond through dehydrationand condensation with a hydroxy group, an amino group, a carbonyl group,a silanol group, and the like on the surface of a solid support, forexample. The polymerization reaction of the polymerizable monomer can becarried out according to the above.

The polymer can be adsorbed on or chemically bonded to the surface ofthe insoluble support by applying the polymer to the surface of theinsoluble support. Examples of the coating method include known methodssuch as a method for preparing a polymer solution, and immersing thesupport therein or spraying the polymer solution thereto. After thepolymer is applied thereto, it is preferable to dry it at roomtemperature or through heating. If the polymer is chemically bondedthereto, reactions may be carried out under the corresponding reactionconditions. Accordingly, the polymer layer is formed on the surface ofthe insoluble support.

The “carrier for concentrating a glycan” is obtained by introducing achain transfer group such as a mercapto group onto the surface ofinorganic particles whose surfaces have hydroxy groups, such as silicabeads serving as the insoluble supports, and synthesizing the polymerlayer having a betaine structure thereon. First, the chain transfergroups are introduced on the surface of inorganic particles using asilane coupling agent having a chain transfer group. At this time, thesilanol group generated through hydrolysis of a hydrolyzable group suchas an alkoxy group of the silane coupling agent and a hydroxy group onthe surface of inorganic particles undergo dehydration and condensationto form a covalent bond, and thus the chain transfer group isintroduced. Then, the inorganic particles provided with the chaintransfer group and a (meth)acrylic monomer in which at least some of themonomers have a betaine structure are radically polymerized throughaddition of a polymerization initiator in an appropriate solvent. Thechain transfer group introduced on the inorganic particles serve as thepolymerization initiation point, the surface of inorganic particles iscoated with a polymer layer having a betaine structure, and thus thepolymer layer is formed.

A purifying agent and the glycan-containing sample obtained in Step (I):the glycan-containing sample acquisition step are brought into contactwith each other. At this time, the glycan-containing sample may bedesalted or the like using a known method. Because the purifying agenthas increased hydrophilicity, glycans can be specifically adsorbedthereon due to hydrophilic interaction, and a large number of peptidefragments and the like that are present in the sample are not adsorbedon the purifying agent and remain in a free state.

The glycan retention force of the purifying agent is proportional to theconcentration of the organic solvent. Therefore, an organic solvent or amixed solvent of an organic solvent and water can be used as a solventof the reaction solution for adsorbing glycans on the purifying agent.The solvent can be selected as appropriate depending on the type ofglycan to be concentrated or the like. There is no particular limitationon the organic solvent as long as glycan can be dissolved therein, andexamples thereof include acetonitrile, tetrahydrofuran, acetone,dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, and1-butanol. Preferably, organic solvents such as 1-butanol and ethanolare suitably used. It is possible to use various buffer solutions inorder to adjust the pH. Also, if a mixed solvent of an organic solventand water is used, the mixing ratio of the organic solvent and water is3:1 or more and 1000:1 or less in terms of volume ratio, for example.

The purifying agent on which glycans are adsorbed can be cleaned asneeded. It is possible to remove impurities such as proteins, peptidefragments, lipids, and salts other than the glycans adsorbed on thepurifying agent through cleaning. It is possible to use theabove-described solvent as a cleaning liquid, for example.

Step (III)/Glycan Elution Step

Step (III) is a step of eluting the glycans from the purifying agent onwhich the glycans have been adsorbed in Step (II): the glycan adsorptionstep.

It is possible to use an organic solvent or a mixed solvent of anorganic solvent and water as an eluent for eluting the adsorbed glycansfrom the purifying agent, and to select the eluent as appropriateaccording to the types of glycan to be eluted and purifying agent. Theabove-described solvent can be used as the organic solvent. Also, it ispossible to efficiently elute glycans by using a highly hydrophilicsolvent in this step. It is also possible to use water without using anorganic solvent, and to use a mixed solvent of an organic solvent andwater, for example. If a mixed solvent is used, the volume ratio of theorganic solvent to water is 3 or less, for example, and particularlypreferably, only water can be used without using an organic solvent.

There are no limitation on Step (II): the glycan adsorption step andStep (III): the glycan elution step, and some or all of the steps may beperformed using a batch method, a spin column method, or the like.Although the batch method and the spin column method will be describedlater in detail, the reagents, reaction conditions, and the like are asdescribed above.

Batch Method

If a batch method is used, the glycans are adsorbed on the purifyingagent by bringing the glycan-containing sample obtained in Step (I): theglycan-containing sample acquisition step and the purifying agent usedin this embodiment into contact with each other in an appropriatecontainer (e.g., a microtube, a centrifuge tube, a microplate, or thelike) (Step (II)). At this time, the purifying agent is preferablyimmobilized on the insoluble support. Then, the purifying agent-glycancomplex is subjected to solid-liquid separation, and the liquid phaseportion containing impurities such as proteins, peptide fragments,lipids, and salts is removed, and only the complex is collected.Solid-liquid separation can be performed through spontaneoussedimentation by gravity, centrifugation, or the like, and the liquidphase portion can be removed through suction or the like. Also,solid-liquid separation may be performed through accumulation of complexusing magnetic force by adding a magnetic material such as ferrite tothe insoluble support for this carrier. In that case, centrifugation orthe like need not be performed. Also, solid-liquid separation may beperformed through filtration by passing a mixture through a filter, andat this time, it may be performed under vacuum pressure or underpressure.

Then, the purifying agent on which glycans are adsorbed is cleaned. Itis possible to remove impurities such as proteins and peptide fragmentsother than the glycans adsorbed on the purifying agent through cleaning.Cleaning can be performed by immersing the purifying agent on which theglycans are adsorbed in a cleaning liquid in an appropriate container,and repeating the replacement of the cleaning liquid. The purifyingagent on which the glycans are adsorbed is introduced into anappropriate container, a cleaning liquid is added thereto, the resultingmixture is shaken or stirred, operation for removing the liquid phaseportion through solid-liquid separation is repeated, and thereby thepurifying agent can be cleaned, for example. Solid-liquid separation canbe performed as described above.

After cleaning is performed, the glycans are eluted from the purifyingagent on which the glycans are adsorbed (Step (III)). The glycans can beeluted by immersing the purifying agent in the eluent. After thecleaning liquid is sufficiently removed, an appropriate amount of theeluent is added to the carrier on which the glycans are adsorbed, andthe resulting mixture is shaken or stirred, for example. Then, thecarrier is collected through solid-liquid separation, and the eluate iscollected in a new appropriate container (e.g., a collection tube or acollection plate, etc.). Solid-liquid separation can be performed asdescribed above. The glycans can be concentrated through distillation ofthe eluate as needed.

Spin Column Method

If a spin column method is used, a container having a built-in filter orthe like, such as a filter cup, may be used. It is possible to use afilter cup having opening portions at the upper portion and the lowerportion thereof, and the lower opening portion is covered with a filter,for example. If a filter cup is used, the glycan-containing sampleobtained in Step (I): the glycan-containing sample acquisition step isintroduced in the filter cup filled with the purifying agent used inthis embodiment, and the purifying agent and the sample are brought intocontact with each other in the reaction solution by passing the samplethrough the filter cup. The purifying agent is preferably immobilized onthe insoluble support. The sample may be passed through free fall due togravity and centrifugation, or the like, or may be passed under vacuumpressure or under pressure. After the sample has passed therethrough,the discharged liquid, which contains free proteins, peptide fragments,lipids, salts, and the like that have passed through the purifyingagent, is removed.

Then, the purifying agent on which glycans are adsorbed is cleaned. Itis possible to remove impurities such as proteins, peptide fragments,lipids, and salts other than the glycans adsorbed on the purifying agentthrough cleaning. Cleaning can be performed by passing the cleaningliquid through the purifying agent in the filter cup, and cleaning canbe continuously performed from adsorption of glycans. The cleaningliquid can be passed as described above.

After cleaning is performed, the glycans are eluted from the purifyingagent on which the glycans are adsorbed. The glycans can be continuouslyeluted by passing the eluent through the purifying agent in the filtercup through cleaning operation from adsorption of glycans. The eluentthat has passed through the purifying agent is collected in anappropriate container (e.g., a collection tube, a collection plate, orthe like). The eluent can be passed as described above. The glycans canbe concentrated through distillation of the eluate as needed.

Step (IV)/Glycan Labeling Step

Step (IV) is a glycan labeling step of reacting a glycan labelingreagent in a glycan labeling solution and the glycans with each other,and the method for preparing a glycan from a glycoprotein of thisembodiment may include Step (IV) as needed.

The “glycan labeling solution” contains at least a “glycan labelingreagent”, and may contain water, a buffer solution, and/or an organicsolvent, or the like. The glycan labeling solution may have ultravioletabsorption characteristics or fluorescence characteristics. Due to theabove-described characteristics being provided, it is possible toperform detection using an analyzer, such as LC-MS.

There is no particular limitation on the “glycan labeling reagent” aslong as it has a reactive group for a glycan and a modification group tobe attached to the glycan. Examples of the reactive group for the glycaninclude an oxylamino group, a hydrazide group, an amino group, and anactive ester group. The modification group can be selected by thoseskilled in the art as appropriate according to a method for analyzing aglycan.

If the glycan labeling reagent has an amino group as a reactive groupfor a glycan, it is possible to use a compound having an amino grouphaving ultraviolet absorption characteristics or fluorescencecharacteristics as the glycan labeling reagent, for example. In such acompound having an amino group, examples of the modification group to beattached to the glycan include an aromatic group. When a labelingcompound having an amino group and an aromatic group is used,modification is performed through reductive amination. Because thearomatic group has ultraviolet-visible absorption characteristics andfluorescence characteristics, the aromatic group is preferable due todetection sensitivity in UV detection or fluorescent detection beingimproved.

Specific examples of the labeling compound that provides such anaromatic group include 8-aminopyrene-1,3,6-trisulfonate,8-aminonaphthalene-1,3,6-trisulphonate, 7-amino-1,3-naphtalenedisulfonicacid, 2-amino9(10H)-acridone, 5-aminofluorescein, dansylethylenediamine,2-aminopyridine, 7-amino-4-methylcoumarine, 2-aminobenzamide,2-aminobenzoic acid, 3-aminobenzoic acid, 7-amino-1-naphthol,3-(acetylamino)-6-aminoacridine, 2-amino-6-cyanoethylpyridine, ethylp-aminobenzoate, p-aminobenzonitrile, and7-aminonaphthalene-1,3-disulfonic acid.

In particular, the compound having an amino group may contain2-aminobenzamide. 2-Aminobenzamide may be preferable because2-aminobenzamide is less likely to be influenced by impurities such asproteins, peptide fragments, lipids, and salts even in a large reactionscale. Note that derivatives of the above-described compound are alsopreferably used as long as the function of the labeling compound can bemaintained.

If the glycan labeling solution contains a buffer solution, examples ofa buffer agent include ammonium carbonate, ammonium hydrogen carbonate,ammonium chloride, diammonium hydrogen citrate, and ammonium carbamate.Although there is no particular limitation on the pH of the buffersolution, the pH thereof is preferably 5 to 10.

If the glycan labeling solution contains an organic solvent, examples ofthe organic solvent may include one or more selected from the groupconsisting of an aprotic polar organic solvent, a protic polar organicsolvent, and an aprotic non-polar organic solvent. Specific examples ofthe organic solvent include aprotic polar organic solvents such asdimethylsulfoxide (DMSO), dimethylformamide (DMF), andN-methylpyrrolidone (NMP), protic polar organic solvents such as organicacids (formic acid, acetic acid, propionic acid, butyric acid, and thelike) and alcohols (methanol, ethanol, propanol, and the like), andaprotic non-polar organic solvents such as hexane. These solvents may beused alone or in combination of two or more.

From the viewpoint of more preferably obtaining the effect of shorteningthe time required for the glycan labeling step, it is possible to use anorganic acid such as formic acid, acetic acid, propionic acid, orbutyric acid as an organic solvent. In particular, from the viewpoint ofperforming operation with ease, it is possible to use acetic acid as anorganic acid.

If the boiling point of the protic polar organic solvent is relativelylow (e.g., if the boiling point is less than 140° C.), in addition to aprotic solvent, a solvent whose boiling point is higher than that ofthis protic solvent may be used in combination. Accordingly, it ispossible to reduce the volatilization rate of the above-described proticpolar organic solvent having a relatively low boiling point in theglycan labeling step. As a result, it is possible to suppress undesiredprecipitation of unreacted substances in the glycan labeling step.Accordingly, it is possible to obtain a labeled glycan in high yield. Itis possible to select a mode in which such a solvent with a higherboiling point (referred to as a high boiling point solvent hereinafter)is used in combination, in a case where the scale of glycan is small, ina case where the amount of solvent is small, and/or, in a case where thereaction time is long.

An example of the high boiling point solvent is an aprotic polar organicsolvent having a boiling point of 140° C. to 200° C. Specific examplesof the high boiling point solvent include aprotic polar organic solventssuch as dimethylsulfoxide, dimethylformamide, and N-methylpyrrolidone.

If an aprotic polar organic solvent serving as a high boiling pointsolvent is used in combination, from the viewpoint of improving thesolubility and reactivity of 2-aminobenzamide, which is a labelingcompound, and a reducing reagent, the amount thereof is preferably lowerthan that of the protic polar organic solvent in terms of vol %, and maybe 4 vol % or more and less than 100 vol %, and may be 4 vol % to 70 vol%.

The “glycan labeling step” may be performed before Step (III): theabove-described glycan elution step, or after Step (III). Therefore, thereaction between the glycan labeling reagent and the glycans in theglycan labeling solution may be performed in a state in which theglycans are adsorbed on the purifying agent, or in a state of being theeluate eluted from the purifying agent. Also, the glycans may be labeledin Step (III), that is, while the glycans are being eluted from thepurifying agent on which the glycans are adsorbed. Furthermore, theglycan labeling solution may be used as the above-described “eluent foreluting the adsorbed glycans from the purifying agent”.

The temperature of reaction between the glycan labeling reagent and theglycans in the glycan labeling solution may be 4° C. or more and 80° C.or less, for example, and may be 25° C. or more and 70° C. or less, forexample. The reaction temperature is preferably the lower limit orhigher because the reaction time is shortened, and the reactiontemperature is preferably the upper limit or lower because partialdecomposition of glycans due to high temperatures is suppressed. Also,the reaction time in the glycan labeling step may be 5 minutes or moreand 600 minutes or less, for example, and may be 30 minutes or more and300 minutes or less, for example. The reaction time is preferably thelower limit or higher in terms of quantitative labeling, and thereaction time is preferably the upper limit or lower because partialdecomposition of glycans due to high temperatures is suppressed.

Note that portions of the free glycans acquired in Step (I): theglycan-containing sample acquisition step are of an aldehyde type, whichform a glycan oxime. With a conventional technique, if an aldehydegroup, which is a functional group of a free glycan, is reduced and thefree glycan is converted to alditol, the glycan cannot be directlylabeled. Also, if the aldehyde group, which is the functional group of aglycan, is converted to hydrazone by hydrazine, the glycan needs to bereturned to the original free glycan through acetylation operation againin order to provide the label.

In contrast, according to Step (I): the glycan-containing sampleacquisition step, it is possible to obtain glycans released as glycanoximes that can be directly labeled. That is, the glycans released fromthe glycoprotein contain glycan oximes, and the glycan oximes can bedirectly labeled. Therefore, the free glycans obtained in Step (I) canbe obtained in a solution as a mixture of glycosylamine, glycan oxime,and a normal glycan having a hemiacetal hydroxy group at the reducingend, and these can be collectively labeled.

Step (V): Reduction Step

Step (V) is a reduction step of causing a reaction with a reductionsolution containing a reducing reagent, and if the method for preparinga glycan from a glycoprotein in this embodiment includes Step (IV): theglycan labeling step, the method may also include Step (V): thereduction step as needed.

In modification through reductive amination, a Schiff base, which isformed through a reaction between the aldehyde group formed at thereducing end of the glycan and the amino group of the labeling compound,is reduced using a reducing reagent, and thereby a modification group isintroduced at the reducing end of the glycan, which enables efficientlabeling, for example.

The “reduction solution” contains at least a “reducing reagent”, and maycontain water, a buffer solution and/or an organic solvent, and thelike.

The “reducing reagent” may contain at least one selected from the groupconsisting of sodium cyanoborohydride, sodium triacetoxyborohydride,methylamine borane, dimethylamine borane, trimethylamine borane,picoline borane, and pyridine borane, for example. Highly safe labelingis possible by using less toxic picoline borane.

From the viewpoint of both safety and reactivity, it is preferable touse picoline borane (2-picoline-borane). From the same viewpoint, ifpicoline borane is used as a reducing reagent, it is preferable to use2-aminobenzamide as the glycan labeling reagent used in Step (IV): theglycan labeling step described above, for example.

The final concentration of the “reducing reagent” may be set to aconcentration range of 1.0 mmol/L or more and 250 mmol/L or less, andmay be preferably set to a concentration range of 1.2 mmol/L or more and239 mmol/L or less. The efficiency of labeling glycan is improved byoptimizing the concentration of the reducing reagent in this range.

If a buffer solution and/or an organic solvent is added to the reductionsolution, it is possible to use the buffer solution and/or the organicsolvent used in Step (IV) described above in the same manner. Note that,if picoline borane is used as a reducing reagent, it is preferable thata solvent contains a protic polar organic solvent. This makes itpossible to dissolve picoline borane at a high concentration and thus toshorten the time required for the step. A mixed solvent of a proticpolar organic solvent such as acetic acid and an aprotic polar organicsolvent such as dimethylsulfoxide may be used as a solvent.

The “reduction step” may be performed in any stage as long as theproduct resulting from the reaction between the glycans and the glycanlabeling reagent can be reduced. A glycan reducing reagent may be addedto the glycan labeling solution, and the reduction step may be performedsimultaneously with Step (IV): the glycan labeling step, for example.

Kit for Preparing Glycan from Glycoprotein

A kit for preparing a glycan from a glycoprotein according to thisembodiment is a kit suitable for carrying out the method for preparing aglycan from a glycoprotein according to the embodiment described in thesection “Method for preparing glycan from glycoprotein” above, the kitincluding:

(a) a hydroxylamine compound;

(b) a basic reagent; and

(c) a purifying agent for purifying a glycan having a length ofmonosaccharide or more, the purifying agent being composed of a compoundhaving a betaine structure. It is possible to provide reagents requiredfor preparing a glycan from a glycoprotein, as a kit in this manner.Furthermore, this kit may contain (d) a glycan labeling reagent, and maycontain (e) a reducing reagent. It is possible to prepare a glycan froma glycoprotein more easily by assembling reagents, information, and thelike required for preparing the glycan from the glycoprotein into a kit.

The “kit” may include a protocol for using this kit as an instructionmanual. The instruction manual may be written or printed on paper orother media, or may be recorded on an electronic medium such as amagnetic tape, a magnetic disk, or an optical disk.

Furthermore, the “kit” may include reagents and containers required forcarrying out this kit. Examples thereof include a cleaning liquid forcleaning, and an eluent for eluting glycans from a purifying agent onwhich the glycan are adsorbed. The reagents included in this kit may beprovided in the form of lyophilized powder, and in that case, the kitmay further include a dilution liquid for dilution when the reagents areused. Also, the kit may include containers such as filter cups,multi-well plates, filter plates, and microtubes, and the containers maybe filled with the reagents included in this kit. The definition of eachterm and preferred embodiments are as described in the section “Methodfor preparing glycan from glycoprotein” above.

Device for Preparing Glycan from Glycoprotein

A device for preparing a glycan from a glycoprotein according to thisembodiment is a device suitable for carrying out the method forpreparing a glycan from a glycoprotein according to the embodimentdescribed in the section “Method for preparing glycan from glycoprotein”above, the device including:

a first container holding portion configured to hold a first containerin which a glycoprotein-containing sample that contains the glycoproteinis accommodated; a reagent introducing portion configured to introduce areagent into the first container; and a second container holding portionconfigured to hold a second container that includes a purifying agentfor preparing a glycan having a length of a monosaccharide or more, thepurifying agent being composed of a compound having a betaine structure,

in which the reagent introducing portion includes a glycan releasingreagent introducing portion configured to introduce a glycan releasingreagent that contains a hydroxylamine compound and a basic reagent intothe first container. It is possible to prepare a glycan from aglycoprotein more easily by assembling reagents, members, and the likerequired for preparing the glycan from the glycoprotein into a device.

Note that the configuration of the device, which will be describedlater, is an example of the device for preparing a glycan from aglycoprotein according to this embodiment, and the scope of the presentinvention is not limited by this configuration. Also, the definition ofeach term and preferred embodiments are as described in the section“Method for preparing glycan from glycoprotein” described above.

The “first container holding portion” is for holding the first containerin which a glycoprotein-containing sample is accommodated. There is noparticular limitation on the mode in which the first container holdingportion holds the first container, and examples thereof include a modein which most of the first container is fitted and held in a holdingopening or a holding hole in the first container holding portion, forexample. In addition, examples thereof include a mode in which anengaging recess (engaging protrusion) in the first container is engagedwith and held by an engaging protrusion (engaging recess) of the firstcontainer holding portion, and a mode in which the first container issandwiched and held by a sandwiching portion of the first containerholding portion.

The “second container holding portion” is for holding the secondcontainer that contains a purifying agent for purifying a glycan havinga length of monosaccharide or more, the purifying agent being composedof a compound having a betaine structure. There is no particularlimitation on the mode in which the second container holding portionholds the second container, and the second container holding portion canhold the second container in the same manner as the first containerholding portion.

The “reagent introducing portion” is for introducing a reagent into thefirst container held by the first container holding portion. The reagentintroducing portion includes at least a glycan releasing reagentintroducing portion that introduces a glycan releasing reagent thatcontains a hydroxylamine compound and a basic reagent into the firstcontainer. The hydroxylamines and the basic reagent may be mixedtogether in the glycan releasing reagent introducing portion to preparethe glycan releasing reagent, or they may be mixed together in advanceto prepare the glycan releasing reagent and then be introduced into theglycan releasing reagent introducing portion.

The “reagent introducing portion” may be able to introduce a reagentinto the second container held by the second container holding portion,and include a glycan-containing sample introducing portion forintroducing a glycan-containing sample obtained by releasing glycansfrom the glycoprotein from the first container, a cleaning liquidintroducing portion for introducing a cleaning liquid for cleaning thepurifying agent, and an eluent introducing portion for introducing aneluent for eluting the adsorbed glycan. Also, the reagent introducingportion may include the glycan labeling reagent introducing portion thatintroduces a glycan labeling reagent into the first container and/or thesecond container. These may be configured as separate and independentcomponents from the glycan releasing reagent introducing portion, or maybe configured as the same constituent member.

There is no particular limitation on the mode in which the reagentintroducing portion introduces a reagent, and examples thereof include amode in which liquid is sent from a liquid sending source in which theliquid to be sent is stored into the first container via a tubularmember. In addition, examples thereof include a mode in which the liquidcollected in the tubular member is injected into the first container.

Furthermore, a “solid-liquid separation portion” for subjecting thecontent of the second container to solid-liquid separation may beprovided. The “solid-liquid separation portion” separates a solid and aliquid in the content contained in the second container. Here, the solidis substantially the purifying agent and the glycans adsorbed on thepurifying agent.

If the solid-liquid separation portion is provided, a container providedwith a filter that is capable of solid-liquid separation (e.g., a filtercup, a filter plate, or the like) may be used as the second container.Furthermore, a collection container (e.g., a collection tube, acollection plate, or the like) may be mounted on such a container, andthen the collection container may be used. Also, in this case, thesecond container holding portion may be configured to include acollection container holding portion for holding the collectioncontainer mounted on the second container.

There is no particular limitation on the specific separation method ofthe solid-liquid separation portion, and any one of centrifugalfiltration, filtration under vacuum pressure, and filtration underpressure may be used. The solid-liquid separation portion may beconfigured as a constituent member independent of the second containerholding portion. In this case, a container transfer portion forautomatically transferring the second container from the secondcontainer holding portion to the solid-liquid separation portion may beincluded. The container transfer portion may be configured to transferonly the second container in the transfer of the second container, or totransfer the second container in a state in which a collection containeris mounted on the second container. The container transfer portion maybe configured to include an arm that operates to hold and open thesecond container directly or indirectly (i.e., via the collectioncontainer) and move the second container, and an arm control portionthat controls the operation of the arm.

Also, a temperature adjustment portion may be included, and if thetemperature adjustment portion is included, the temperature adjustmentportion need only have at least a heater function. The temperatureadjustment portion heats the first container and/or the second containerto at least a temperature required to release a glycan, make a glycan beadsorbed on a purifying agent, and/or elute the glycan from thepurifying agent on which the glycan is adsorbed.

Also, at least any or preferably all of the operable components (e.g.,the reagent introducing portion, the arm, the solid-liquid separationportion, and the temperature adjustment portion) may be automaticallycontrolled. Accordingly, it is possible to prepare glycans fromglycoproteins more quickly.

EXAMPLES

The following specifically describes the present invention usingexamples. However, the present invention is not limited to theseexamples.

Example 1 Synthesis of Purifying Agent

A carrier for concentrating a glycan in which a polymer whose side chainhaving a betaine structure is bonded to the main chain thereof isimmobilized on an insoluble support was synthesized as the purifyingagent. Although the synthesis of a polymer that contains aconstitutional unit derived from 2-methacryloyloxyethylphosphorylcholine (referred to as an “MPC polymer” hereinafter) on thesurface of silica beads is described specifically, it is not intended tolimit the scope of the present invention.

Synthesis of Purifying Agent

Introduction of Chain Transfer Group into Silica Beads

5 g of a silane coupling agent having a chain transfer group was addedto a liquid mixture of 50 mL of an aqueous solution of acetic acid witha pH of 3.0 and 50 mL of ethanol, the resulting mixture was stirred atroom temperature for 1 hour to hydrolyze the silane coupling agent, 5 gof silica beads, which are inorganic particles, were introduced as anexample of an insoluble support, and the resulting mixture was stirredat 70° C. for 2 hours, the silica beads were collected from the reactionsolution through suction filtration, and was heated at 100° C. for 1hour. Then, the silica beads were dispersed in ethanol, the resultingmixture was shaken well, and the supernatant was removed throughcentrifugation and dried.

Synthesis of Polymer

2-Methacryloyloxyethylphosphorylcholine (manufactured by NOFCORPORATION, and referred to as “MPC monomer” hereinafter), which willbe the constitutional unit of the polymer was dissolved in ethanol toproduce 20 mL of 0.8 mol/L monomer solution. Then, AIBN was added torealize 0.027 mol/L and stirred until the resulting mixture becameuniform. Then, 4 g of silica beads provided with the above-describedchain transfer group was introduced therein, and was reacted at 70° C.for 6 hours in an argon gas atmosphere. Then, the silica beads werecollected from the reaction solution through centrifugation, weredispersed in ethanol, were shaken well, were collected through suctionfiltration, and were dried to obtain a carrier in which the polymercontaining a constitutional unit derived from2-methacryloyloxyethylphosphorylcholine was immobilized on silica beads(simply referred to as “carrier” hereinafter).

Check Physical Properties of Purifying Agent

Measurement of Weight of Layer Containing Polymer that is Introduced onSurface of Carrier

The weight of the layer containing the polymer that was introduced onthe surface of the above-described carrier was obtained by measuring therate of weight loss obtained by increasing the temperature from roomtemperature to 500° C. at increments of 10° C./min in an air atmosphereusing a TGA device (TG/DTA6200 manufactured by Seiko Instruments Inc.)and maintaining 500° C. for 1 hour. When the weight of the layercontaining the polymer that was introduced on the surface of particleswas calculated based on this value and the surface area of the particlesper unit weight separately obtained using the BET method, the weight was1.08 mg/m².

Example 2 Relationship between reducing reagent concentration and yieldwhen labeling glycans

Reaction Condition 1 (1) Preparation of Glycan Labeling Solution

(1-1) A mixed solvent of 10% acetic acid, 45% methanol, and 45% waterwas prepared.(1-2) 1 M 2-aminobenzamide and picoline borane, which was a reducingreagent, were dissolved in the solution (1-1) so as to realize aconcentration of 0.6, 1.2, 2.3, 4.7, 9, 47, and 239 mM.

(2) Release of O-Linked Glycan

Bovine fetuin protein (20 μg) was added to a liquid mixture of 50%hydroxylamine aqueous solution and DBU (5:2 (volume ratio), 15 μL) andwas mixed, and the resulting mixture was heated using a heat block at37° C. for 75 minutes.

(3) Collection of O-Linked Glycan

1000 μL of acetonitrile was added to 15 μL of the solution (2) preparedin (2) above and was mixed well, and the resulting mixture was added to1 mg of the carrier synthesized as the purifying agent for purifyingglycans in Example 1 above, and was mixed well. The mixture was added toa spin column (Ultrafree-MC, Millipore Cat#: UFC30VNB), and the carrierand the solution were separated from each other through centrifugationusing a table-top centrifuge. Then, 400 μL of acetonitrile was added,and the solution was removed through centrifugation. 400 μL ofacetonitrile was added again, and the solution was removed throughcentrifugation.

(4) Labeling of O-Linked Glycan

50 μL of the solution (1-2), which was a liquid mixture of picolineborane and 2-aminobenzamide that was prepared to have the concentrationsdescribed in (1-2), was added to the carrier, the solution was collectedand was mixed with the above solution, and thereby the glycans werefluorescently labeled through a reaction at 50° C. for 2.5 hours

(5) Purification of O-Linked Glycan

The solution (4) containing the fluorescent labeled glycan obtained in(4) above was applied to a cleanup column (BS-45403 accessory availablefrom Sumitomo Bakelite Co., Ltd.) to remove an excess reagent, and thefluorescently labeled glycans were analyzed through HPLC.

Reaction Condition 2

The reaction proceeded in the same manner, except that picoline boranewas dissolved in the solution (1) so as to realize a concentration of47, 93, 239, 374, 561, 748, and 935 mM in Reaction Condition 1 above.

Results

The graph in FIG. 2 shows the results of Reaction Condition 1, and thegraph in FIG. 3 shows the results of Reaction Condition 2. Thehorizontal axis indicates the picoline borane concentration [mM] and thevertical axis indicates the peak total surface area, that is, thelabeling efficiency with 2-aminobenzamide.

From the results shown in FIGS. 2 and 3, it was confirmed that thelabeling efficiency was the highest when the concentration range ofpicoline borane was 5 mM or more and 20 mM or less, and the yieldfurther decreased at a concentration of higher than 239 mM. The reducingreagent is usually used at a concentration of 200 mM or more, forexample, and sodium cyanoborohydride, which is a reducing reagent thatis widely used in this technical field, is usually used at aconcentration of 1 M (1000 mM), for example. Therefore, it can beunderstood that the results obtained in this example are very lowconcentrations, compared to the concentrations of reducing reagent usedin the technical field.

Example 3 Preparation of Glycan/Purifying Agent (1) Release of O-LinkedGlycan

Bovine fetuin protein (20 μg) was added to a liquid mixture of 50%hydroxylamine aqueous solution and DBU (5:2 (volume ratio), 15 μL) andwas mixed, and the resulting mixture was heated using a heat block at37° C. for 75 minutes.

(2) Collection of O-Linked Glycan

1000 μL of acetonitrile was added to 15 μL of the solution (1) preparedin (1) above and was mixed well, and the resulting mixture was added to1 mg of the carrier synthesized as the carrier for purifying glycans inExample 1 above, and was mixed well. The mixture was added to a spincolumn (Ultrafree-MC, Millipore Cat#: UFC30HVNB), and the carrier andthe solution were separated from each other through centrifugation usinga table-top centrifuge. Then, 400 μL of acetonitrile was added, and thesolution was removed through centrifugation. 400 μL of acetonitrile wasadded again, and the solution was removed through centrifugation.

(3) Labeling of O-Linked Glycan

50 μL of the liquid mixture of picoline borane and 2-aminobenzamide wasadded to the carrier on which the treatment in (2) above was performed,the solution was collected and was mixed with the above solution, andthereby the glycans were fluorescently labeled through a reaction at 50°C. for 2.5 hours.

(4) Purification of O-Linked Glycan

The solution (3) containing the fluorescent labeled glycan obtained in(3) above was applied to a cleanup column (BS-45403 accessory availablefrom Sumitomo Bakelite Co., Ltd.) to remove an excess reagent, and thefluorescently labeled glycans were analyzed through HPLC.

Comparative Example 4 Preparation of Glycan/Cleanup Column (1) Releaseof O-Linked Glycan

O-linked glycans were released in the same manner as (1) of Example 3.

(2) Collection of O-Linked Glycan

1000 μL of acetonitrile was added to 15 μL of the solution prepared in(1) above and was mixed well, and the resulting mixture was added to acleanup column (BS-45403 accessory available from Sumitomo Bakelite Co.,Ltd.), which is the carrier for purifying glycans used in thiscomparative example, and the solution was removed through centrifugationusing a table-top centrifuge. Then, 400 μL of acetonitrile was added,and the solution was removed through centrifugation. 400 μL ofacetonitrile was added again, and the solution was removed throughcentrifugation.

(3) Labeling of O-Linked Glycan

50 μL of the liquid mixture of picoline borane and 2-aminobenzamide wasadded to the cleanup column, the solution was collected and was mixedwith the above solution, and thereby the glycans were fluorescentlylabeled through a reaction at 50° C. for 2.5 hours.

(4) Purification of O-Linked Glycan

O-linked glycans were purified in the same manner as (4) of Example 3.

Comparative Example 5 Preparation of Glycan/Graphite Carbon Column (1)Release of O-Linked Glycan

O-linked glycans were released in the same manner as (1) of Example 3.

(2) Collection of O-Linked Glycan

1 mL of acetonitrile was passed through a graphite carbon column(Supelclean ENVI-Carb C available from Sigma-Aldrich Co., LLC). 3 mL ofwater was then passed therethrough. Then, 15 μL of the solution preparedin (1) above and 180 μL of 0.1% acetic acid water were mixed togetherand was passed through a graphite carbon column, which is the carrierfor purifying glycans used in this comparative example. 3 mL of waterwas passed therethrough to wash graphite carbon. A 0.22 μm-filter wasmounted, 500 μL of 50% acetonitrile aqueous solution containing ammoniumacetate was passed therethrough, and the solution was collected. Thecollected solution was dried using a centrifugal evaporator.

(3) Labeling of O-Linked Glycan

50 μL of the liquid mixture of picoline borane and 2-aminobenzamide wasadded to the sample dried in (2) above, and the glycans werefluorescently labeled through a reaction at 50° C. for 2.5 hours.

(4) Purification of O-Linked Glycan

O-linked glycans were purified in the same manner as (4) of Example 3.

The graphs in FIGS. 4 and 5 show the results of Example 3 andComparative Examples 4 and 5. FIG. 4 shows a chart of the results ofHPLC analysis. FIG. 5 shows the summary of the results of HPLC analysisin bar graphs, the horizontal axis showing the types of carriers usedfor purification, and the vertical axis showing the peak total surfacearea, that is, labeling efficiency with 2-aminobenzamide. As a result,when the purifying agent of the present invention synthesized in Example2 is used, O-linked glycans were efficiently prepared (Example 3). Onthe other hand, when a cleanup column was used, the yield was about thehalf the yield obtained when the purifying agent of the presentinvention was used (Comparative Example 4), and when graphite carbon wasused, most of the glycans was not collected (Comparative Example 5).

INDUSTRIAL APPLICABILITY

The present invention provides a method, a kit, and a device forpreparing a glycan from a glycoprotein. Therefore, the present inventioncan be used in the technical fields in which preparation of glycans fromglycoproteins, in particular, preparation of glycans that includeO-linked glycans with a small molecular weight, is required, forexample, such as life science, medical care, and drug discovery, toelucidate a mechanism of the development of various diseases associatedwith structural changes in glycans, and to develop disease treatments,diagnostic techniques, and the like.

1. A method for preparing a glycan from a glycoprotein, comprising: (I)a step of obtaining a glycan-containing sample by bringing a glycanreleasing solution that contains a hydroxylamine compound and a basicreagent into contact with the glycoprotein and releasing glycan from theglycoprotein; (II) a step of adsorbing a glycan having a length of amonosaccharide or more on a purifying agent for purifying the glycan bybringing the glycan-containing sample into contact with the purifyingagent, comprising a compound having a betaine structure; and (III) astep of eluting the glycan from the purifying agent.
 2. The methodaccording to claim 1, further comprising (IV) a glycan labeling step ofreacting a glycan labeling reagent in a glycan labeling solution and theglycan with each other.
 3. The method according to claim 2, furthercomprising (V) a reduction step of reducing the glycan with a reductionsolution that contains a reducing reagent.
 4. The method according toclaim 3, wherein a concentration of the reducing reagent in thereduction solution is 1.0 mmol/L or more and 250 mmol/L or less.
 5. Themethod according to claim 1, wherein the glycan releasing solutioncontains hydroxylamines in an amount of 2% or more and 70% or less. 6.The method according to claim 1, wherein the hydroxylamine compound isat least one selected from the group consisting of hydroxylamine, saltsof hydroxylamine, O-substituted hydroxylamine, and salts ofO-substituted hydroxylamine.
 7. The method according to claim 1, whereinthe basic reagent is at least one selected from the group consisting ofalkali metal hydroxides, weak acid salts of alkali metals, alkalineearth metal hydroxides, alkaline earth metal salts dissolved in aqueousammonia, and organic bases.
 8. The method according to claim 7, whereinthe alkali metal hydroxide is lithium hydroxide, sodium hydroxide, orpotassium hydroxide, the weak acid salt of alkali metals is sodiumbicarbonate or sodium carbonate, the alkaline earth metal hydroxide iscalcium hydroxide, barium hydroxide, or strontium hydroxide, thealkaline earth metal salt is calcium acetate, calcium chloride, bariumacetate, or magnesium acetate, and the organic base is1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5,7-triazabicyclo[4.4.0]dec-5-ene,7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene,1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetrametylguanidine,or cetyltrimethylammonium hydroxide.
 9. The method according to claim 1,wherein the purifying agent contains the compound having the betainestructure as an active ingredient.
 10. The method according to claim 9,wherein the purifying agent is a carrier for concentrating a glycan inwhich a polymer whose side chain that has the betaine structure isbonded to a main chain thereof is immobilized on an insoluble support.11. A kit for preparing a glycan from a glycoprotein, comprising: (a) ahydroxylamine compound; (b) a basic reagent; and (c) a purifying agentfor preparing a glycan having a length of a monosaccharide or more, thepurifying agent comprising a compound having a betaine structure. 12.The kit according to claim 11, further comprising (d) a glycan labelingreagent.
 13. The kit according to claim 11, wherein the hydroxylaminecompound is at least one selected from the group consisting ofhydroxylamine, salts of hydroxylamine, O-substituted hydroxylamine, andsalts of O-substituted hydroxylamine.
 14. The kit according to claim 11,wherein the basic reagent is at least one selected from the groupconsisting of alkali metal hydroxides, weak acid salts of alkali metals,alkaline earth metal hydroxides, alkaline earth metal salts dissolved inaqueous ammonia, and organic bases.
 15. The kit according to claim 14,wherein the alkali metal hydroxide is lithium hydroxide, sodiumhydroxide, or potassium hydroxide, the weak acid salt of alkali metalsis sodium bicarbonate or sodium carbonate, the alkaline earth metalhydroxide is calcium hydroxide, barium hydroxide, or strontiumhydroxide, the alkaline earth metal salt is calcium acetate, calciumchloride, barium acetate, or magnesium acetate, and the organic base is1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5,7-triazabicyclo[4.4.0]dec-5-ene,7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene,1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetrametylguanidine,or cetyltrimethylammonium hydroxide.
 16. A device for preparing a glycanfrom a glycoprotein, comprising: a first container holding portionconfigured to hold a first container in which a glycoprotein-containingsample that contains the glycoprotein is accommodated; a reagentintroducing portion configured to introduce a reagent into the firstcontainer; and a second container holding portion configured to hold asecond container that contains a purifying agent for purifying a glycanhaving a length of a monosaccharide or more, the purifying agentcomprising a compound having a betaine structure; wherein the reagentintroducing portion includes a glycan releasing reagent introducingportion configured to introduce a glycan releasing reagent that containsa hydroxylamine compound and a basic reagent into the first container.17. The device according to claim 16, wherein the reagent introducingportion further includes a glycan labeling reagent introducing portionconfigured to introduce a glycan labeling reagent.
 18. The deviceaccording to claim 16, further comprising a solid-liquid separationportion configured to subject a content of the second container tosolid-liquid separation.